Project Summary/Abstract Diabetes has reached epidemic proportions in the United States and globally, and impaired diabetic wound healing is a significant and growing clinical problem. The long-term goal of our research is to develop small molecule therapeutics to effectively promote healing of diabetic wounds. Diabetic wounds are deficient in stromal derived factor-1??(SDF-1?), a potent chemokine involved in progenitor cell recruitment, angiogenesis, and granulation tissue formation, mediated through binding to the CXCR4 receptor and the establishment of a chemotactic gradient. Our previous research data confirmed that targeting SDF-1?/CXCR4 signaling pathway has great potential to improve diabetic wound healing. Therefore, screening small molecule agonists that can activate CXCR4 receptor and its downstream pathway will provide a novel therapy for diabetic wound healing, and has great potential for clinical application and commercialization. In our previous RO1 (R01DK105010, Identifying CXCR4 receptor agonists to improve diabetic wound healing), we screened the entire NIH SMR library of >370k molecules using our robust testing funnel and identified 303 lead compounds. These lead compounds were further validated with by counter-screen, secondary chemotaxis/migration assays, and their ability to correct abnormal expression of microRNA-146a, 15b, and 29a, resulting in the identification of 2 lead scaffolds. In an exciting preliminary study we tested the ability of our lead scaffold CAG1 to improve diabetic wound healing following injection into murine diabetic wounds. We found that a single CAG1 injection resulted in a significant improvement in the rate of diabetic wound closure, but the optimal formulation and mechanisms of correction remain to be determined. The objective of this work is to determine the ability of our novel small molecule CXCR4 agonists to correct the diabetic wound healing impairment in vivo, optimize the formulation and pharmacokinetics in vitro and in vivo using a medicinal chemistry approach, determine the mechanisms of action, and then extend these observations to the clinically relevant porcine model. The following Specific Aims are proposed: Specific aim 1: Conduct lead optimization of the 5-aryl oxazole and triazolo thiadiazine series using structure- activity- relationship medicinal chemistry approaches, Determine compound in vitro EC50 values in primary and functional assays, KD values in a CXCR4 binding assay and determine effect on human diabetic fibroblast expression of miR15b, and miR29a. Specific aim 2: Perform in vitro stability, skin permeability and metabolism studies and determine the mechanisms of diabetic wound healing correction in a murine model of wound healing for compounds advancing to tier 4. Specific aim 3: Identify a CAG1 or CAG2 lead compound in tier 5 and Validate that CXCR4 agonist corrects the diabetic wound healing impairment and is non-toxic in a pre-clinical porcine model.